JP3121611B2 - Gene vector for expressing nerve growth factor in eukaryotic cells - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing nerve growth factor (β-NG) from transformed cells.
By a method for obtaining polypeptides called F), more precisely by recombinant DNA technology using a gene construct insertable into a suitable eukaryotic cell line, the biologically active human mature form (β- Subunit).

A. Nerve Growth Factor (NGF) Nerve growth factor (NGF) was first discovered in mouse sarcomas [Levi-Montalcini, R. et al., J. Ex.
p.Zool. 116: 321,1951], followed by male mouse salivary submandibular gland [Varon, S. et al., Biochemistry 6: 2202,196].
7], and snake venom [Angeletti, R.
H.) Proc. Natl. Acad. Sci., USA 65: 668, 1970]. Other sources that are relatively rich in NGF include guinea pig prostate [Harper, G. et al.
Nature 279: 160,1979] and the human placenta [Goldstein, LD, et al., Neurochem. Res. 3:
175, 1978, and Life Scie by Walker, P. et al.
nce 26: 195, 1980, Fidia Patent 47745A88]. Small amounts of NGF have been found in other tissues, such as the mammalian central nervous system [Varon, S., D.
iscussions in Neuroscience, Vol. II, No. 3, 1985, and Hefti, F. et al., Neuroscience 14:55, 1985]. Although the physiological relationship between these sources of NGF and the apparent site of action is unclear, NGF is secreted from various epithelial tissues that require innervation from cells that respond to NGF. It is generally considered.

Usually, NGF obtained from mouse salivary submandibular gland is used to test NGF in vitro and in vivo. The range of in vitro biological activities of NGF is
Determined in both primary neurons and cloned cell lines. The primary neurons that respond to NGF in vitro include fetal sensory neurons from the spinal ganglion root (fetal days 8-12), autonomic noradrenergic neurons from the sympathetic ganglia, and developing embryos There are cholinergic fetal neurons from the septum and chromaffin adrenal cells. While sensory and autonomic nervous system neurons depend on NGF for their survival and development, cholinergic neurons not only survive but also differentiate, i.e., express phenotypic properties that connect with neurotransmitters. Does not seem to require NGF either. NG in chromaffin adrenal cells (derived from neural crest) in early development
When F is added, a neural phenotype is expressed. Some cell lines that respond to NGF in vitro include, as described in the literature, chromaffin adrenal cells derived from neural crest tumors, so-called chromaffin cell tumor cells (PC12) and human neuroblasts. There are cell tumor cells. β-NGF
, The cells change their behavior and transition from an intense growth phase to a cell division state.

Nerve growth factor obtained from mouse salivary submandibular gland is also well characterized by chemical and immunochemical profiles. NGF from murine glands acts like a 7S-type protein complex (molecular weight about 140,000 daltons) composed of three subunits (α, β, γ) coordinated with a Zn ⁺ atom.

The most interesting part of the biological activity of this 7S molecule is constructed from two polypeptide chains, each formed from 118 amino acids with a molecular weight of 13,250. Each of its chains or monomers has three sulfide bridges that form a covalent bond between two cysteine residues, which confers strong stability to the tertiary structure of the protein. NGF each bound by weak binding
Form a dimer with a molecular weight of 26,500. Its biological activity is so-called 2.5S or usually β
-Has been shown to be associated with dimers called subunits. It is not known whether this is also present in the monomer.

The gene encoding this β-subunit of NGF (β-NGF) could be identified by genetic engineering techniques [Scott, J. et al., Nature 302: 538,1.
983, Ullrich, A. et al., Nature 303: 821,19.
83, European Patent Publication No. 0 121 338]. The human gene encoding this molecule is located on the short arm of chromosome I and encodes the synthesis of a molecule larger than the biologically active molecule with a molecular weight of 26,500. Thus, the gene initially directs the synthesis of larger NGF precursors or pro-NGF. In addition, β of NGF
-Genes encoding subunits have been proven to be highly conserved in various species ranging from birds to humans [Meier, R. et al., EMBO J. 5: 1.
489,1986].

The elucidation of the nucleotide sequence of murine, human, bovine and chicken β-NGF has made it possible to compare the conserved and non-conserved sites of these molecules with their relationship to biological activity and antigenicity . β-NGF
The overall preservation during the onset period is surprisingly high. Mature NGF purified from the salivary submandibular gland of male mice
Of the 118 amino acids in bovine β-NGF, 19 in chicken β-NGF and human β
-NGF differs by only 11 and only 6 amino acids differ between bovine and human β-NGF. All cysteine residues are exactly conserved in all species. Reduction of the three SS bridges of β-NGF results in complete loss of its biological activity. At first glance, the overall high level of conserved amino acid sequence and the low cross-reactivity of the immunochemical forms are contradictory, but they are in a "cluster" where the amino acid variation between species is specific. It is derived from being arranged in. Hydropathic trac
ings) can demonstrate that these variations are present in almost all hydrophilic sites that are considered to be strong antigenic determinants. It has been found that in all species of NGF tested so far, only one hydrophilic region is strictly conserved.

B. Recombinant DNA Method A series of vectors capable of expressing a target protein in large amounts can be constructed by the recombinant DNA method. According to this approach, molecular biologists are likely to create hybrid molecules that can produce the protein of interest.
DNA sequences can be combined. This method involves various reactions, such as restriction enzyme digestion, ligation of the resulting fragments by ligase, chemical synthesis of the oligonucleotides to be assembled, and available in various laboratories in the art. [Maniatis, T.]
Et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Laboratory
Spring Laboratory, New York, 1982]. To obtain high levels of expression, the DNA element to be assembled must have substantial information. For example, the origin of replication, selectivity for antibiotics, expression promoters, activators of transcription of the gene of interest, and other properties known to the researcher of the material. In combining these elements in a suitable manner to obtain a plasmid, the gene of interest is inserted naturally in terms of regulatory sequences for transcription and translation, and the resulting plasmid is defined by expression. In this way, a plasmid, ie, an expression vector, can express a protein in a host cell. Next, the protein may be obtained by a certain purification method. Elements (promoters) that naturally control the expression of many genes, such as growth factors,
The above expression is not very strong and is only activated under appropriate natural conditions, often unknown. For this purpose, a promoter of known activity is used, for example a series of papovaviruses, or other known promoter gene sequences. Thus, the elements used for high levels of expression are DNs of various origins (eukaryotes, bacteria, viruses, etc.) constructed for the purpose of joining various gene parts to form a hybrid.
It is a combination of A. The transcription and translation activity of a gene depends on the appropriate distance between regulatory and coding sequences.

One of the best ways to properly manipulate the regulatory sequences during this transfer is to place the transferred gene at the same position as in the native gene. One system used is one in which the regulatory sequence also contains some amino acids of the coding sequence. The conjugate with this transgene (union) leads to the fusion protein. However, higher biological values can be obtained if the fusion moiety is removed. Without this fusion protein approach, the usual approach for obtaining genes located in close proximity to regulatory sequences depends on the presence of appropriate restriction sites at which they can be cloned. When a compatible site is not present nearby but at another site, an oligonucleotide or linker containing a desired restriction site may be synthesized to prepare a conjugate of a segment having the same.
If there are no nearby restriction sites where the linker can be used, use a technique to delete the DNA using Bal 31 or S1. In this case, since the deletion cannot be performed accurately, it is necessary to always perform sequencing of various clones to confirm which one is optimal. These systems are very limited for molecular biologists and ultimately need to pioneer alternatives, such as the polymerase chain reaction (PCR), that have a role as emerging techniques. Saiki) et al. Science 239: 487,1
988, and Science of Scharf, SJ 233: 10
76,1986].

According to this approach, the gene segments can be amplified up to 10 ^6. The principle is based on the use of two oligonucleotides that can be paired, each of which is one of the DNA strands to be amplified. The distance of the two oligonucleotides to the investigated gene sequence determines the size of the molecule that can be produced. These two oligonucleotides are constructed so that restriction sites for subsequent cloning are present inside their sequence. This restriction site is naturally occurring or specially constructed by denaturing a minimum number of bases. This method, which can be defined as a site-specific mutation, allows molecular biologists to create restriction sites at theoretically determined positions. On the other hand, creating a site that is compatible with other gene segments facilitates cloning, but in particular also introduces the possibility of binding various targeted gene segments. This approach can be defined as cloning based on direct mutation. In fact, recombinant DNA methods allow direct heterologous expression of a heterologous polypeptide by direct mutation, or expression of a heterologous polypeptide fused to an amino acid sequence portion of a similar polypeptide. be able to. In general, the products obtained by this method are not biologically active [GB Patent Application Publication No. 2007676A, benzene (We
nzel), American Scientist 68,664,1980].

The ability to isolate the human gene for the β-subunit of nerve growth factor offers important possibilities. Producing sufficient quantities of this rare protein is possible by using recombinant DNA technology. Indeed, nerve growth proteins are acceptable as clinically usable for treating various neuropathic diseases. In this sense, there is a literature relating to obtaining the β-subunit of NGF by recombinant DNA technology [EP-A 0 121 388, Bruce
Ce., G.) et al., Neurobiology of Aging 10: 89,1989, Hu, G., et al., Nucleic Acid Research 70: 57,1988, Edwards, RH, Mol. Cell. Biol. 8: 2456. , 198
8. Proc.Natl.Acad.Sci.8 of Enforces (P.)
6: 4756, 1989]. In production, only if it cannot be expressed inexpensively in microbial cells,
Select mammalian cells rather than bacteria. Although it is actually much more economical to produce a specific protein in a bacterial cell line such as E. coli, this host / vector system generally allows only the linear sequence of the amino acids that make up the protein to be accurately reproduced. And provide it as a kind of insoluble mass in bacteria. In the case of certain small molecules, such as interferons or some animal growth proteins, the molecules can be correctly folded in vitro, and in such cases, E. coli can use this material in a manner where certain products are economically advantageous. Would be the system of choice. These systems are used when generally associated with proteins having a single disulfide bond and for peptides or proteins that have uses (as diagnostic antigens or vaccine components) that do not require a well-defined conformation The case is the most productive one.

In therapeutic proteins to which nerve growth factor belongs,
To be active and usable, the correct conformation is required, and there is a need for the absence of an antigenic response. This method of preparation would include glycosylation for proteins obtained from the recombinant DNA, generation of correct disulfide bonds, and other post-transduction modifications. The bacterial cell line, E. coli, cannot meet this requirement, and mammalian eukaryotic cells and yeast can. These problems should be taken into account when applying human β-NGF obtained by biotechnology as a pharmaceutically active substance.

The activity of NGF is dimer, ie 2 of 118 amino acids.
It has been shown to be derived from a combination of two similar polypeptides. When reduced by mercaptoethanol, its biological activity drops to virtually zero. There have been attempts to combine cysteine to regenerate three disulfide bridges, but from a statistical point of view,
Only one-in-one chances of obtaining a correctly structured molecule that is equivalent to the structure corresponding to the natural product. E.co finally obtained
The molecules in li cannot guarantee structural homology and their application to humans as pharmaceutically active substances. In fact, even after purification to homogeneity, human NGF produced from E. coli is not a biologically active dimer in immunoblotting with a polyclonal antibody specific for murine β-NGF. A series of unattributed bands is observed. In addition, this mixture of structures and forms has been shown to be 10-fold less biologically active than similar human forms purified from natural sources, such as placental tissue.

This method of cloning and obtaining nerve growth factor in E. coli involves a series of inaccuracies that can produce secondary effects when administered in vivo, even if the protein of interest is produced at high expression levels. Molecules, for example, antibodies, are produced that recognize and inhibit the biological activity of a naturally occurring molecule. At the same time, E.co
Cloning of the mature molecule in li presents a starting methionine that cannot be removed and is definitely immunogenic because it is located on the exposed portion of the molecule.

Other methods involve cloning prepro-NGF in eukaryotic cells, and utilize the attack of certain peptidases naturally present in eukaryotic cells to obtain mature molecules. Specifically, cloning is performed using Chinese hamster ovary (CHO). Distributed literature indicates that the human NGF from which the entire genome has been cloned has not yet been sequenced, but that the gene has been extended to more than 10 kDa [Ullrich, A.] Nature 303: 821,198
3]. Since such a long gene cannot be normally cloned, it is performed separately from the whole genome sequence. The method contains only the encoded part of this protein
This is for cloning a part of cDNA. Currently, human
Although the complete cDNA for NGF has not been isolated (some sequences lack the 5 'portion), much information is known about NGF messengers of other origins (mouse, cow, chicken, etc.). EMBO [Meier, R. et al.
J. 51489,1986, Selby (HJ) J of Neuron
Research 18: 293,1987], which can lead to interesting inferences. The mouse NGF gene is present in a single copy and produces at least four different messengers of different dimensions [Selby (Se
Ml. Cell. Biol. 7: 3057, 1987]. These different sizes are reflected in particular in the different AUG start codons, -187 and 1 in the mature protein.
21st place is the most important. These messengers are present in various tissues with corresponding varying abundances. In the submandibular gland, messengers starting at -187 are 10 times more abundant than those starting at -121. However, due to various evidences, it is expressed in the brain
It has been shown that NGF Messenger has the most constant percentage because it uses an AUG of exactly -121.

SUMMARY OF THE INVENTIONThe present invention relates to an expression vector in which a regulatory site and a site encoding a β-subunit protein of human NGF are separated by a natural distance, so that the expression vector exists in a natural sequence. Can obtain a mature form of the β-subunit of human nerve growth factor (hβ-NGF) in the culture medium without the presence of one or more amino acids fused to the polypeptide, such as CHO. The expression vector for use in a eukaryotic cell line allows for human NGF
And a method for obtaining the β-subunit. The polypeptide thus obtained exhibits biological activity when used on appropriate target cells.

The hβ-NGFs described herein are used to maintain neurological function and prevent its loss, as well as chronic or acute forms of pathology, cerebrovascular, infectious, inflammatory, compressive,
It can also be used to restore neurodegenerative and immune system modulation late in an acute illness, such as a metabolic disorder.

The present invention is further directed to a genetic construct that can be used for cell transfection, such that it can be implanted in vivo.
Some of these constructs are capable of specifically producing, at the local level, the active form of human growth factor depending on the function of the diet to which they are administered, thus allowing the gene to be under control .

The present invention is further directed to a transformed cell line containing the vector and a culture thereof producing hβ-NGF. The present invention also relates to the neurotrophic factor β
A pharmaceutical preparation containing as active substance one or more novel conjugates of a unit with a natural ganglioside or a derivative or semi-synthetic homolog or a salt thereof.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the hydropathicity of polypeptides of the nerve growth factor β-subunit of human origin.
athicity).

FIG. 2 schematically shows the construction of a pMSGphNGF expression vector.

 FIG. 3 is a schematic diagram of a pMSGphNGF expression vector.

FIG. 4 schematically shows construction of a pSV40MTphNGF expression vector.

FIG. 5 is a schematic diagram of a pSV40MTphNGF expression vector.

FIG. 6 schematically shows the construction of a pSV40phNGF expression vector.

 FIG. 7 is a schematic diagram of a pSV40phNGF expression vector.

FIG. 8 schematically shows construction of a pSV40hNGF expression vector.

DETAILED DESCRIPTION OF THE INVENTION From the above evidence, the human β-NGF of the present invention was cloned exactly starting from the methionine at -121. Analysis of the hydropathic profile showed that the amino acids between -121 and -104 functioned as a lead peptide, indicating that the protein could be secreted. This profile is called Hop
p) et al., Proc. Natl. Acad. Sci., USA 78: 3824, 1981, a hydropathic profile of a polypeptide of the subunit of nerve growth factor of human origin, This is shown in FIG. The position shown as −121 / −104 represents the lead peptide, and the position shown as + 1 / + 118 represents the amino acid sequence of the nerve growth factor β-subunit polypeptide of human origin [Ullrich ) No N
ature 303: 821,1983]. There are certain peptidases for obtaining mature proteins, which are capable of obtaining amino acid sequences from +1 to +118 corresponding to biologically active peptides. This peptidase contained in cells that normally synthesize NGF was shown to also be contained in AT-20 cells. In fact, by introducing a vector containing the vaccinia virus prepro-NGF of murine origin into these cells, they become mature NGF
To produce a 14 kDa molecule in a gel under denaturing and reducing conditions [Mol. Cell. Bio, Edwards, RH].
l.8: 2456, 1988].

We have described the function of the foregoing experience in obtaining the β-subunit of the nerve growth factor described above in one or more of the following ways in a revolutionary way that can be used in eukaryotic cells. Vector constructs were compared. PC
Using the R method, prepro-NGF is cloned with sites engineered to be compatible with various expression vectors, and their restriction sites are as close to the natural distance as possible between regulatory and coding sequences. In a situation completely different from the gene constructs already described in the literature on the expression of the β-NGF subunit. British Technology [British Te
chnologry Ltd., Oxford, United Kingdom], a modified mature human NGF (hβ-NGF) sequence whose restriction site has been created to allow subsequent mutations was combined with a portion of prepro-NGF. . For the purposes of the present invention, the presence of these restriction sites should not be taken as limiting, nor should the origin of the genes be considered as limiting.

Starting from this assumption, we next turn to SV40 (simian virus 40), MTTV (mouse mammary tumor virus), hMTIIa
Prepro-hβ-NGF was cloned under the control of various promoters such as (human metallothionein IIa). Next, these gene sequences introduced into the plasmid were
The cells were transfected, and the obtained cells were proved to be capable of producing biologically active h-NGF in the culture medium. This prepro-NGF has been demonstrated to be essential for the assembly of this molecule, which allows only the β-subunit to be expressed, and indicates that the entire gene construct is considered correct. .

A. General method used The DNA strand is attacked with restriction enzymes according to the manufacturer's instructions. In general, 1 μg of plasmid
Cleave with 1 unit of enzyme in 20μ. The temperature and the incubation time vary depending on the enzyme used, but are generally 37 ° C. for 1 hour. After incubation, the plasmid and gene segments were transferred to 40 mM Tris / HCl, 20 mM
mM sodium acetate, in 1 mM EDTA, agarose gel LMP Agarose [BRL, USA] to give then Geneclean ^TM (GENECLEAN ^TM) kit [BIO 101 Inc., La Jolla, California, USA] from the agarose using Elute. To perform a copy reaction at the 5 'end,
DNA at 15 ° C using 10 units of polymerase (Klenow)
For 15 minutes. As ligase, T4 ligase is used at a concentration of 1 unit per 0.5 μg of DNA in 20 μg of the reaction at 13 ° C. for 12 hours.

For analysis to confirm the correct sequence in the plasmid, HB101 cells are transformed and transformants are selected on agarose plates in LB (Luria Bertani) medium containing 50 μg / ml ampicillin antibiotic. The resulting plasmid contained in HB101 was grown in LB at 100 μg / ml ampicillin and quenched by Qiagen [DIAGE
N GmbH, Düsseldorf, West Germany] using kits to make small or large preparations. An expression vector is prepared from bacterial cells by the Qiagen method.

D for PCR reaction from human placenta in the perinatal phase as follows
Prepare NA. Cut out 0.4 cm ³ pieces of chorion with scissors,
50 mM Tris / hydrochloric acid pH 7.8, 100 mM EDTA, 100 mM NaCl, 1
Suspend in 700μ% SDS. Then 35 μl of proteinase (K 100 μg / ml) is added to this and incubated at 55 ° C. overnight. Then 20 μl of the solution is added to 13 μg / ml RNAse A and incubated for a further 2 hours. Prepare two extracts using phenol and two extracts using chloroform. Then, one volume of isopropanol is added and the DNA is deposited on a glass capillary. At this point, pass through 70% and 100% ethanol and dry. While slowly stirring the obtained DNA in a test tube, a buffer solution (10 mM Tris / HCl pH7.
4. Dissolve in 1mM EDTA). After a few hours, the dissolved DNA is prepared for gene amplification. DNA is usually used to perform PCR
0.1 μg is sufficient.

CHO cells (CCL) according to the manufacturer's procedure (GIBCO) or by the calcium phosphate method
61) and lack of dehydrofolate reductase gene (DHFR
^-) a modified CHO based on transfecting a liposome.

A.2 Transfection of liposomes Cells grown in α-MEM containing 5% fetal calf serum were displaced by trypsinization the day before transfection and reached 70-80% of total growth the next day. To be. Plasmid DNA to be transfected in the density of water 50.mu., diluted to DNA10myug, then it Lipofectin ^TM (Lipofectin ^TM) (Gibco)
Add 50μ and put the whole in a polystyrene tube. After 15 minutes, the resulting mixture is added to cells previously washed with OPTIMEN medium (Gibco). The cells thus obtained are incubated for 8 hours, and then normal medium containing fetal calf serum is added and continued to grow.

A.3 Transfection Method to Obtain Stable Transformants Using Calcium Phosphate
Two-fold concentrated BBS (2 × BBS) and 0.25M CaCl ₂ . 2 × BBS is prepared as follows: 50 mM N, N-bis-2- (hydroxyethyl) -2-aminoethanesulfonic acid [Calbiochem], 280 mM NaCl,
And 1.5 mM Na ₂ HPO ₄ were dissolved in water, the pH was adjusted to 6.5,
The whole was then filtered through 0.45 μm, and simultaneously with 2.5 M CaCl ₂
Prepare 10 × CaCl ₂ as a solution of

Cells are seeded with 5 × 10 ⁵ cells / 10 cm plate / 10 ml of growth medium and incubated at 35 ° C. overnight. Plasmid 20μg
Is mixed with 0.5 μM of 0.25 M CaCl ₂ and 0.5 ml of 2 × BBS. This mixture is incubated at ambient temperature for 15 minutes. The resulting mixture was then impregnated into the medium, all of which was incubated at 35 ° C. in 3% carbon dioxide overnight. In order to obtain stable transformed cells having the vector of the present invention, a vector expressing hβ-NGF, pSV2Neo
Co-transfection is performed by adding the vector at a ratio of 10: 1. Two days after this transfection, the resulting cells are trypsinized, plated at a 10-fold lower concentration compared to this transfection, and transformed using 1 mg / ml G418 neomycin sulfate (Gibco) into stable transformants. Start selecting immediately. The transformants are then analyzed for integration of the gene construct by Southern blot.

A.4 Expression For expression, Han's F12 / DME H-21 containing 15 mM Hepes and 10% fetal calf serum is maintained in culture medium for all cells. To purify hβ-NGF, the medium is changed every three days and replaced with fresh serum-free medium.

B. Preferred Embodiments B.1. Description of Construction of Expression Vectors Three different vectors are prepared: the first and the second are regulatory elements, MMTV (mouse mammary tumor virus) and hMTIIA (human metallothionein IIa, respectively). ) Are two vectors that can be chemically derived. In this method, the hβ-NGF gene can be expressed under control. In contrast, in the third vector,
hβ-NGF is cloned under the control of the SV40 (simian virus 40) promoter.

B.2. Cloning in pMSG expression vector The pMSG vector was obtained from Pharmacia [Upsala, Sweden]. According to the manufacturer's instructions, the gene to be introduced must be inserted into the multiple cloning site between the Nhe I and Sal I restriction sites.
This gene is translated by its first AUG. In this case, the nerve growth factor is under the control of the MMTVLTR (mouse mammary tumor virus long terminal repeat) promoter. The activity of this promoter can be induced by administering a glucocorticoid, such as dexamethasone. In this transcription, splicing occurs with the small t-antigen of SV40 and polyadenylation occurs with the long t-antigen of SV40. This plasmid also contains the bacterial gene for xanthine guanine phosphoribosyltransferase (xgpt) which is used to select for stably transformed CHO KI cells.

In utilizing the PCR technique to allow the cloning of prepro-NGF in this plasmid, restriction sites immediately before the starting AUG were used, so that the distance between the regulatory sequences and the coding sequence of prepro-NGF was as natural as possible. Create Synthesize two oligonucleotides: the first between bases 9122 and 9147 (Ulrich Nature 303: 821, 1983) should have the following sequence: The base before the starting AUG is mutated as described above, and then a synthetic oligo having the following sequence is made: This oligonucleotide is called (Xba I). A second oligonucleotide containing bases between 9521 and 9342 (Ulrich's Nature 303: 821,1983) is complementary to this sequence that can be subjected to PCR and has the following sequence: This oligonucleotide contains an EcoR I site inside that is capable of binding prepro-NGF to mature NGF. This oligonucleotide is called (EcoRI).

Using an oligonucleotide synthesizer, these two oligonucleotides were converted to a 330B DNA synthesizer [Applied
According to the standard procedure of Applied Biosystems, USA], it is synthesized in a solid phase by the phosphoramidite method. These are (a) treated in ammonia at 55 ° C. for 12 hours, (b) dried in a vacuum centrifuge, and (c)
Resuspend in 2.5 M ammonium acetate, (d) precipitate with 3 volumes of cold ethanol (−20 ° C.), and (e) rewash with 80% cold ethanol and resuspend in water. The concentrations of the two oligonucleotides are analyzed by spectroscopy. Perkin Elmer Cetus DNA Termal Cy
cler Amplificator], and use the reagents of the relevant DNA ^™ amplifier [Perkin Elmer-Cetus] kit for the amplification. Briefly, a mixture containing 200 μM of each oligonucleotide, 0.5 μM each of dATP, dTTP, dCTP, dGTP oligonucleotide, and 0.1 μg of human DNA, and
100 μl in total mixture containing 0.5 units of TAQ polymerase
Of the reaction buffer, all coated with paraffin oil to prevent evaporation. In the case of human DNA, the amplification reaction is performed by operating the apparatus at 35 cycles. In both cases, the cycle is as follows: 1 minute at 94 ° C, 2 minutes at 45 ° C.
Min, 72 ° C for 3 min. The 300 bp amplified fragment was prepared using the Geneclean ^™ kit [BIO 101 Inc., La Jolla,
By melting agarose using California, USA, low melting agarose [NuSiev
e)] Purified in gel. Xbai and EcoR
Cleavage with I restriction enzyme and repurification as described above. The fragment thus purified is cloned into the pGEM4 vector [Promega] at the Xbai and EcoRI sites. The resulting plasmid is called pGEM4Xba-NGF.

This plasmid (pGEM4Xba-NGF) was replaced with Hind III-EcoR
Cleavage at the I site, purifying the resulting 300 bp fragment,
It is cloned into the HindIII-EcoRI site of the pUC18BBG26 vector [British Biotechnology, Ltd. (British Biotechnology Ltd.), Oxford, UK]. pUC18BBG26 contains the gene for the cassette that constitutes hβ-NGF, i.e., inside its sequence is a non-naturally occurring restriction site capable of replacing the determined domain, in other words a mutation Restricted sites that can be used have been created. The resulting vector is called pUC18hNGFC. Cut this vector with BamHI,
The extension is copied 5 'by Klenow polymerase. It is cut at the XbaI site and the resulting 760 bp fragment is purified by agarose gel. This fragment is cloned between the Nhe I and Sma I sites of the pMSG expression vector. A summarized scheme for obtaining this vector, called pMSGphNGF, is shown in FIG. 2, and a schematic diagram of the vector is shown in FIG.

B.3. Cloning of NGF Under the Control of Metallothionein Metallothionein IIa (MT IIa) is Zn ⁺⁺ or Cd ⁺⁺
Alternatively, this vector was prepared because it can be induced by other heavy metal ions and thus its expression can be controlled. In addition, to enhance expression of this promoter, an SV40 enhancer is added to the 5 'end of the promoter of MT IIa, while the small t-antigen of SV40 and polyA are always used for splicing and polyadenylation. First, the metallothionein promoter is linked to prepro-hβ-NGF as follows: Plasmid (phMTIIA) is replaced with the restriction enzymes HindIII and BamH.
The metallothionein promoter was isolated by digestion with I [Karin, H. Nature 299: 797,1].
982], the resulting 841 bp fragment containing the promoter was ligated into the pGEM4 vector [Promega M.
adison), WI, USA] at the HindIII-BamHI site. This vector is called pGEM4hMTIIa. This promoter part is 3 'with some natural codons.
It extends to the side and contains the first codon of AUG methionine, immediately followed by a BamHI restriction site. hMT II
A BamHI restriction site was created immediately after the first AUG so that the gene could be brought into phase so that preprohβ-NGF could be cloned under the control of this promoter by using the first methionine of the promoter of a. Bases 9133 and 9160 [Ulrich's Nature 303: 8
21,1983]. Array: Is mutated as in the following sequence: This oligonucleotide is called BamHI. The changed base also includes a change in the amino acid sequence, and in fact, Asp
From Ser and Pro to Met, 2nd and 3rd respectively
Includes the th amino acid change. However, this change has no significant effect on the hydropathicity profile, and the molecule is secreted normally.

This oligonucleotide, together with the EcoRI oligonucleotide, is used to amplify the prepro-NGF described above. The resulting purified fragment was cut at the BamH I and EcoR I sites and then the pGEM4hMTIIa vector BamH I and EcoR
Clone between I restriction sites. PGhMTphNG
Call it F.

To get the SV40 enhancer (activator)
Extract from the PMSGphNGF plasmid as follows. P
The MSGphNGF vector is cut with BamHI and then the extension is copied 5 'using Klenow polymerase. It was then cut with Hind III and the resulting 500 bp
The fragment is cloned between the Nae I and Hind III restriction sites of pGEM3. The resulting vector is called pGSV40.

To obtain an expression vector, the above pieces are ligated in a triple union of fragments as follows: pMSGphNGF
The vector is cut with EcoRI and BamHI restriction enzymes and the resulting 1500 bp fragment is purified. The pGhHTphNGF vector is cut with the HindIII-EcoRI enzyme and the 1100 bp fragment is purified. Together these two fragments are pGSV
Clone between the Hind III and BamHI restriction sites of the 40 vector. A summarized scheme for obtaining this expression vector, called pSV40MTphNGF, is shown in FIG. 4, and a schematic diagram of this vector is shown in FIG.

This vector pSV40MTphNGF is stably introduced into CHO KI cells as a double transfection along with the pSV2Neo plasmid and selected with 1 mg / ml neomycin G418 (Gibco, BRL) as described above. This plasmid is called phMT
Co-transfect with the plasmid and select colonies containing high copy number. In this case, CHO KI cells are exposed to 50 mM zinc sulfate for 24 hours to induce metallothionein synthesis and selection using cadmium chloride starting at a concentration of 2.5 μM and up to 20 μM. Cells containing a high copy number of hNGF are used for expression of this molecule.

B.4.Cloning of NGF under the control of the promoter enhancer of SV40 The pGEM4XbaNF plasmid was cut with Hind III and EcoR I restriction enzymes, and the resulting 300 bp fragment was pSV40MTphNG.
Replace between the Hind III and EcoR I restriction sites of the F vector. A summarized scheme for obtaining this vector, called pSV40phNGF, is shown in FIG. 6, and a schematic diagram of the vector is shown in FIG.

This is a standard construction vector in which regulatory sequences are assigned to the SV40 promoter / enhancer. This vector is co-transfected into CHO KI cells with the pSV2Neo plasmid, and clones producing the nerve growth factor β-polypeptide are analyzed.

Selection in CHO KI dhfr Cells In order to amplify the gene of the aforementioned vector in CHO KI Dfhr ⁻ , the vector is substantially modified by cloning the DHFR + gene upstream from the hNGF gene. PSV2dhfr vector encoding DHFR +
Cleave at the nd III site, then copy the extension at 5 'using a polymerase. Then it is BamH
The resulting 1800 bp fragment was cut into pSV40h
XhoI restriction site in the NGF vector (blunted by polymerase as described above), and clone into BamHI. In this way, a pSV40hNGF expression-selection plasmid is created. The summarized scheme of this vector is shown in FIG.
Shown in the figure.

This plasmid contains no nucleotides,
CHO in alpha MEM medium containing 10% fetal calf serum
Transfect KI cells normally. Two days later, cells are trypsinized to 1/10 of the concentration described above and amplification is performed using MTM (methotrexate) from 10 μM to 500 μM. Cells that survive at the highest concentration are used for hNGF expression.

Determination of Biological Activity After the insertion of one of the three vectors described above, and after stabilizing them, an in vitro test to determine the biological activity in the CHO cell line culture medium was carried out using chromium. Affinity cell tumors are performed on fetal cells PC-12 [Greene LA et al., Rev. Neurosci. 3: 353, 1982]. By using a culture medium in which a non-transformed CHO cell line is growing, or by inhibiting the activity of hβ-NGF present in the culture medium by a polyclonal antibody specific for NGF of murine or bovine origin. Confirm the specificity of this reaction. The three cell lines transformed and stabilized by the individual vectors described above are human
Produces the β-subunit of NGF in its biologically active form.

Pharmaceutical Compositions The hβ-NGF of the present invention can be formulated by known methods for producing pharmaceutically useful compositions. The method of formulating a pharmaceutical composition containing a human NGF molecule (β-subunit) obtained by the recombinant DNA described above and also containing a ganglioside and a phospholipid can be used as a medicament which can be administered to a patient. Known methods for preparing pharmaceutically acceptable compositions can be used to adjust the effective amount of hNGF molecules that can be mixed in the mixture with the pharmaceutically acceptable vehicle.
Suitable vehicles, and their formulations containing other proteins, are described, for example, in "Remington's Pharmaceutical Sciences" [Remingto
n's Pharmaceutical Sciences, Mac Publishing Company, Easton, Pa., USA, 1985]. These vehicles include "deposit formulations" for injection.

Based on these, the pharmaceutical formulations of the present invention include one or more pharmaceutically acceptable agents added to buffer them to the appropriate pH and to make them isotonic with physiological fluids. Solutions of nerve growth factor, or a lyophilized powder thereof, together with a vehicle or diluent that may be used, are not exhaustive. When preparing a lyophilized agent, a supporting excipient such as mannitol or glycinin may be used, and a desired isotonic buffered solution having a desired pH may be prepared using a desired excipient. Prepare a volume of the appropriate buffered solution. As a pharmaceutical solution for a pharmaceutical composition of a molecule of nerve growth factor obtained by recombinant DNA in a desired volume of isotonic solution, a similar solution as described above can be used, Using a physiological buffered solution containing an appropriate concentration of phosphate or citrate to obtain an isotonic pharmaceutical preparation each time at the desired pH, e.g., neutral pH. Not limited to that.

Pharmaceutical formulations of the present invention further include, but are not limited to, suppositories for rectal administration containing a lyophilized excipient, such as a water-soluble, self-emulsifying glycogelatin type or the like. In this preparation, the recombinant DNA
May be present in a content of 0.01% to 1/1% based on the weight of all excipients. The suppository may contain, but is not limited to, an appropriate amount of acetylsalicylate.

The above pharmaceutical preparations can be intended for oral, rectal, parenteral, topical, inhalation and intracerebral application. Thus, it is in a solid or semi-solid form, such as dragees, tablets, gelatinous opercul
a), capsules, suppositories, soft gelatin capsules and the like. For parenteral or intracerebral application, select a dosage form suitable for intramuscular injection, subcutaneous administration, or for intravenous or intracerebral infusion or injection. It therefore comprises a solution of the active substance, and a lyophilized powder of the active substance, which comprises one or more pharmaceutically active substances which are suitable for their use and have a permeability compatible with physiological fluids. Mention may be made of combinations of acceptable excipients or diluents. For topical administration, preparations in the form of creams or ointments are contemplated for use, and for inhalation, preparations of sprays such as nasal sprays are contemplated.

The preparations of the present invention can be administered to humans or animals. Solutions, sprays, ointments and creams preferably contain 0.01% to 10% of the active ingredient, and solid preparations preferably contain 5% to 50% of the active ingredient. The dosage form to be administered depends on the indication, the desired effect and the mode of administration chosen.

The invention further relates to the use of the novel complex of the β-subunit of nerve growth factor NGF and gangliosides or derivatives thereof for the aforementioned indications. The daily dosage of injections (subcutaneous or intramuscular or intracerebral) for humans varies from 0.05 mg to 5 mg of active substance per kg of body weight.

Having described the invention, it is clear that the methods described herein can be modified in various ways.
Such modifications should not be deemed to depart from the spirit and scope of the invention, and all modifications that would be obvious to one skilled in the art are intended to be within the scope of the claims of the invention. .

[Brief description of the drawings]

FIG. 1 is a graph showing the profile of nerve growth factor β-subunit hydropathy of human origin;
FIG. 2 is a schematic diagram showing a construction method of a pMSGphRNGF expression vector, FIG. 3 is a schematic diagram of a pMSGphRNGF expression vector, and FIG. 4 is a schematic diagram showing a construction method of a pSV40MTphNGF expression vector. FIG. 5 is a schematic diagram of a pSV40MTphNGF expression vector, FIG. 6 is a schematic diagram showing a construction method of a pSV40phNGF expression vector, and FIG.
FIG. 8 is a schematic diagram of the expression vector, and FIG.
FIG. 2 is a schematic diagram showing a method for constructing an NGF expression vector.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Alesandro Negro Via Umago, Padua, Italy No. 5 (56) References JP-A-60-84299 (JP, A) JP-A-4-128300 (JP, A) JP-A-2-291270 (JP, A) Neurobiol. Aging, Vol. 10, No. 1 (1989. Jan.-Feb.) P. 89-94 Bio / Technology, Vol. 5, No. 7 (1987) p. 720-725 Gene, Vol. 51, No. 2-3 (1987) p. 197-204 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/12 C12N 5/10 C12P 21/02 C07K 14/48 BIOSIS (DIALOG) WPI (DIALOG)

Claims (10)

(57) [Claims] (1) An array as well as A fragment of human genomic DNA of about 300 base pairs obtained by polymerase chain reaction amplification of human genomic DNA using a primer having the following sequence: (b) a fragment of said 300 base pair fragment (a) encoding a portion of human β-NGF A 460 base pair EcoR I-BamHI fragment fused directly to the 3 'end; human prepro-β- from position -121 to position +118.
A replicable expression vector comprising a fragment of about 760 base pairs of DNA encoding NGF, wherein the 760 base pair fragment comprises Nhe I and Sma I of plasmid pMSG.
A replicable expression vector inserted between the sites. (2) Array as well as A fragment of human genomic DNA of about 300 base pairs obtained by polymerase chain reaction amplification of human genomic DNA using a primer having: (b) a portion of human β-NGF, A 460 base pair EcoR I-BamHI fragment fused directly to the 3 'end; (c) a 500 base pair BamHI containing the SV40 enhancer element.
(D) a 841 base pair HindIII-BamHI fragment containing the promoter of the human metallothionein IIa gene; human prepro-β-NGF at positions -121 to +118
A replicable expression vector comprising a fragment of about 760 base pairs of DNA encoding: SV40 enhancer (c) comprising human metallothionein II
It is operably linked to the 5 'end of the promoter (e) of a, and the 3' end of the promoter is composed of Nae I and pGEM3.
Operably linked to the 5 'end of an approximately 760 base pair DNA fragment encoding human prepro-β-NGF between BamHI.
A replicable expression vector. (3) Array as well as A fragment of human genomic DNA of about 300 base pairs obtained by polymerase chain reaction amplification of human genomic DNA using a primer having: (b) a portion of human β-NGF, A 460 base pair EcoR I-BamHI fragment fused directly to the 3 'end; (c) a 500 base pair BamHI containing the SV40 enhancer element.
(D) a 841 base pair Hind III-B containing the promoter of human metallothionein IIa and the initiator methionine;
amHI fragment; consisting of human prepro-β- from position -121 to position +118
A replicable expression vector comprising a fragment of about 760 base pairs of DNA encoding NGF, wherein the SV40 enhancer (c) comprises human metallothionein IIa
Is operably linked to the 5 'end of the promoter (d), and the 3' end of the promoter is Nae I and Ba of pGEM3.
About 760 encoding human prepro-NGF between the mHI sites
A replicable expression vector operably linked to the 5 'end of a base paired DNA fragment. 4. A recombinant host cell transformed with the expression vector according to claim 1. 5. The cell according to claim 4, wherein said cell is a mammalian cell. 6. The cell according to claim 5, wherein the mammalian cell is a CHO cell. (7) cells Xba I oligonucleotide having the sequence (B) amplifying a 300 base pair DNA fragment by polymerase chain reaction using the XbaI and EcoRI oligonucleotides as primers and human genomic DNA as template; (c) pGEM4 vector The 300 bp DNA fragment was cloned into the XbaI and EcoRI sites of pGEM4Xba-NGF plasmid; (d) Plasmid pGEM4Xba-NGF was transformed with HindIII and EcoRI.
To produce a first 300 bp fragment; (e) cloning the first 300 bp fragment into the pUC18BBG26 plasmid at the HindIII-EcoRI site to form pUC18h
(F) cutting the pUC18hNGFc with BamH I and Xbal,
(G) preparing the 760 base pair fragment with Nhe I and Sma of the pMSG vector;
(H) isolating a fragment containing the metallothionein IIa (MTIIa) promoter by cutting phMTIIa with HindIII and BamHI; (i) cloning into the pMSGphNGF plasmid; The fragment containing the MT IIa promoter was cloned into the Hind III and Bam HI sites of the pGEM vector,
(j) cutting the prepro NGF DNA sequence with BamHI and EcoRI, and cloning the resulting fragment between the BamHI and EcoRI restriction sites of pEM4hMTIIa to obtain pGhMTphNGF; (K) pMSGphNGF was cut with BamHI, the 5 'extension was copied with Klenow polymerase, cut with HindIII, and the resulting 500 bp fragment was digested with NaeI and HindII of pGEM3.
Cloning between the I sites to produce PGSV40; (l) pGhMTphNGF was cut with Hind III and EcoR I to give the first
And a second fragment is prepared by digesting pMSGphNGF with EcoR I and BamH I, and the first and second fragments are digested with pGSV
PS cloned between 40 Hind III and BamHI sites
A method for producing an expression vector encoding human β-NGF, comprising producing V40MTphNGF. 8. (i) transforming a mammalian cell with the plasmid according to any of claims 1 to 3, (ii) culturing the transformed cell; and (iii) producing the transformed cell. And recovering human β-NGF. 9. The method according to claim 8, wherein the mammalian cell is a CHO cell. 10. The plasmid is a recombinant DNA plasmid having the structure of pSV40MTphNGF, the vector comprising: as well as A fragment of human genomic DNA of about 300 base pairs obtained by polymerase chain reaction amplification of human genomic DNA using a primer having: (b) a 300 bp fragment encoding the human β-NGF portion; A 460 base pair EcoR I-BamHI fragment fused directly to the 3 'end of (c) a 500 base pair BamHI containing the SV40 enhancer element.
-Hind III fragment; (d) 841 base pair Hin containing the promoter of human metallothionein IIa gene and initiator methionine
dIII-BamHI fragment; consisting of human prepro-β- from position -121 to position +118
It contains a fragment of approximately 760 base pairs of DNA encoding NGF, and the SV40 enhancer (c) contains human metallothionein IIa
Is operably linked to the 5 'end of the promoter (d), and the 3' end of the promoter is Nae I of pGEM3 and
760 encoding human prepro-β-NGF between the BamHI sites
The method according to claim 8 or 9, which is operably linked to the 5 'end of the base pair DNA fragment.